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WO2000046377A1 - Particules de vecteur retroviral mises au point par genie genetique, capables d'infecter des cellules ne se divisant pas - Google Patents

Particules de vecteur retroviral mises au point par genie genetique, capables d'infecter des cellules ne se divisant pas Download PDF

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WO2000046377A1
WO2000046377A1 PCT/US2000/002852 US0002852W WO0046377A1 WO 2000046377 A1 WO2000046377 A1 WO 2000046377A1 US 0002852 W US0002852 W US 0002852W WO 0046377 A1 WO0046377 A1 WO 0046377A1
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cells
vector
retroviral
snv
cell
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WO2000046377A9 (fr
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Ralph Dornburg
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Thomas Jefferson University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae

Definitions

  • This invention generally relates to the field of virology, and, more particularly, to a system of generating and producing retroviral vector particles from retroviruses which are non-pathogenic for humans and capable of transducing therapeutic genes into non-dividing cells.
  • retroviral vectors used in human gene therapy protocols have been derived from murine leukemia virus (MLV), an amphotropic C-type retrovirus.
  • MLV murine leukemia virus
  • HAV-1 human immunodeficiency virus
  • the present invention describes procedures for the generation and production of safe retroviral vectors derived from C-type retroviruses, which are non- pathogenic for humans and which are capable of transducing genes into specific non-dividing cells. Transduction of genes into non-dividing cells has been achieved in the present invention by using the approaches described herein. Using site- directed mutagenesis, a specific signal (nuclear transportation signal sequence) has been introduced into the matrix protein of the retroviral vector particle. This signal is sufficient to enable the penetration of the nucleus of the non-dividing cell.
  • the introduction of a nuclear localization sequence into the matrix protein adds a new feature to the C-type retroviral vector particle: the capability to actively penetrate the nucleus of a quiescent cell.
  • This system has two major advantages over current vector systems. First, efficient gene transfer into non-dividing cells is achieved. Moreover, this system is, but does not have to be, combined with an existing cell-type-specific gene delivery system. Second, this system is safe, since the retroviral particles used have been derived from a retrovirus which is non- pathogenic in humans.
  • Nuclear translocation sequence means nuclear localization sequence or nuclear transportation signal sequence.
  • the retroviral vector system described here may be used to deliver genes into various tissues of the human body, which consists of non-dividing cells, e.g. , liver, hematopoietic stem cells, brain and many more cell-types. It can be combined with a cell-type-specific gene delivery system developed by us earlier to transfer genes into a very distinctive cell-type only. Thus, there will be numerous human gene therapy applications into various organs transducing a large variety of therapeutic genes.
  • the system described here overcomes the last major hurdle and disadvantage of current retroviral vector systems, which is the incapability to infect non-dividing cells.
  • This invention also relates to a method for preparing particles, which contain genetically modified core proteins involved in the import of retroviral cores into the nucleus.
  • the present invention describes procedures for the generation and production of safe retroviral vectors derived from retroviruses, which are non-pathogenic for humans and capable of transducing genes into non-dividing cells. This has been achieved using the following approaches. Using site-directed mutagenesis, a specific signal (nuclear transportation signal sequence) has been introduced into the core protein of the retroviral vector particle. This signal is sufficient to enable the penetration of the nucleus of the non-dividing cell.
  • the present invention pertains to a C-type retroviral vector particle having a genetically modified MA core protein, wherein a consensus nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type core protein.
  • the present invention pertains to a method for preparing a C-type retroviral particle having the capability to infect quiescent cells which comprises a retroviral vector core particle which contains a genetically engineered MA protein wherein a nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type MA protein.
  • the present invention pertains to a C-type retroviral particle having the capability to infect quiescent cells which comprises a retroviral vector core particle which contains a genetically engineered MA protein, wherein a nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type MA protein, which facilitates nucleus penetration of the infected target cell .
  • the present invention pertains to a method for preparing a C-type retroviral particle having the capability to infect quiescent cells which comprises a retroviral vector core particle which contains a genetically engineered MA protein wherein a nuclear translocation sequence has been created by altering the amino acid sequence of the wild-type MA protein, which facilitates nucleus penetration of the infected target cell.
  • Figure 1 illustrates the principle of a retroviral packaging line derived from a C-type retrovirus.
  • retroviral proteins are expressed from different plasmid DNAs. These RNA transcripts do not contain encapsulation sequences. Thus, they are not encapsulated into retroviral particles.
  • B Such helper cells are transfected with a retroviral vector plasmid construct. The RNA transcript of the retroviral vector contains an encapsulation sequence, and, therefore, is encapsulated into virions supplied by the helper cell.
  • Supernatant tissue culture medium is used to infect fresh target cells, prol and pro2: promoters to express viral protein coding sequences; poly
  • polyadenylation sequence is used to infect fresh target cells.
  • Figure 2 illustrates a consensus nuclear translocation sequence of HIN-1 as well as HIN-1 nuclear translocation sequence of an individual HIN-1 strain (isolated from quiescent human brain cells).
  • S ⁇ N amino acid sequences of S ⁇ N at the some position in MA as in HIN-1 and mutations introduced into this region using site directed mutagenesis.
  • Three mutations generate a nuclear translocation sequence in the MA protein of S ⁇ V.
  • the figure shows all single, double, and triple mutations, termed S ⁇ N-MA-mutl, 2, 3, etc.
  • S ⁇ N-MA-mut7 The effect of each single, double, and the final triple mutations (e.g., S ⁇ N-MA-mut7) has been tested individually for efficiency of replication and the ability to infect quiescent cells.
  • FIG 3. illustrates the plasmids used to express retroviral protein or to introduce a retroviral vector into packaging cells.
  • An S ⁇ N retrovirus provirus is shown at the top. It has the genomic organization typical for a C-type retrovirus.
  • the retroviral vector pCXL contains the bacterial beta-galactosidase gene in place of the retroviral protein coding sequences.
  • pIM29 is a plasmid vector for the expression of the S ⁇ V wild-type envelope gene. The env gene is expressed from the murine leukemia virus (MLV) U3 promoter (MLV-U3-pro). Polyadenylation is mediated by the simian virus 40 (SV40) polyadenylation signal sequence (SV40polyA).
  • SV40 simian virus 40
  • SV40polyA polyadenylation signal sequence
  • pTC53-7A5zeo expresses a chimeric single chain antibody-envelope (scA-Env) fusion protein.
  • the scA recognizes a cell surface protein expressed on various human hematopoietic cells, e.g. , T-lymphocytes and macrophages (Engelstadter, M. , et al. Targeting human T-cells by retroviral vectors displaying antibody domains selected from a phage display library, 1999 (submitted for publication).
  • the plasmid construct is similar to pIM29. It contains the adenovirus tripartite leader sequence (AVtl) downstream of the MLN-U3 promoter for enhance gene expression.
  • AVtl adenovirus tripartite leader sequence
  • pRD136 is similar to plasmid pTC53-7A5zeo. It expresses the S ⁇ N wild-type gag-pol proteins. The location of the MA sequence, which has been mutated, is indicated at bottom and compared to the ⁇ LS sequence of HIN-1 (for more details see Figure 2). E: encapsidation sequence.
  • Figure 4 illustrates the protocol used to test whether mutations in the MA protein of a retrovirus would interfere or hamper retrovirus replication and whether the mutations would enable the infection of quiescent cells.
  • a cell line was made which expresses the envelope protein of a retrovirus (e.g. , S ⁇ N) and a retroviral vector transducing the bacterial ⁇ -galactosidase gene (the cell line was termed DSE29-cxl).
  • Transfection of a construct expressing wild-type or mutant Gag-Pol generates a complete retroviral packaging line. Virus was harvested from such cell lines and fresh target cells were infected.
  • FIG. 5 FACS analysis of the D ⁇ A content of D17 or C8166 cells. Examples of flow cytometry patterns of cells stained with propidium-iodide. A: proliferating D17 cells: B: D17 cells growth-arrested with mitomycin for 36 hours. C: proliferating C8166 cells; D: C8166 cells growth arrested with mitomycin for 36 hours.
  • Figure 6 Experiments to test for the presence of viral D ⁇ A in the nuclei of infected quiescent cells.
  • Target cells dog D17 cells, human H9 or Jurkat cells
  • mitomycin mitomycin
  • an aliquot of the cells was subjected to FACS analysis to verify the growth arrest.
  • Four other aliquots were infected with vector virus particles containing wt or mutant (pRD136-m7) MA proteins.
  • Two days after infection another aliquot of the cells was subjected to FACS analysis to verify the maintenance of the growth-arrested state of the cells.
  • D ⁇ A was isolated from the nuclei of one aliquot, another aliquot was stained with X-gal to verify efficient infection.
  • FIG. 7 Infection of human blood monocyte-derived macrophages. Infection with particles containing A: wt-MA and wt-Env; B: wt-MA, wt- + targeting-Env; C: MA derived from RD136-m7 and wt-Env; D: MA derived from RD136-m7, wt- + targeting-Env.
  • This invention relates to genetically engineered retroviral vector particles capable of transducing genes into non-dividing cells.
  • the genetically engineered retroviral vector particle contains a mutant core protein containing a nuclear translocation sequence involved in the active import of the retroviral core (pre- integration complex) into the nucleus of the infected target cell.
  • Retroviruses are wide-spread in nature and are associated with various diseases in animals and man. (Varmus, H. E. and P. Brown. 1988. Retroviruses, p. 53-108. In D. E. Berg and M. M. Howe (eds.), Mobile DNA. American Society for Microbiology, Washington, D.C.). They contain an RNA genome, which is converted into a double-stranded DNA copy. This DNA copy is inserted into the genome of the host cell. Thus, a retrovirus becomes part of the genomic outfit of the infected cell. Some retroviruses carry non-retroviral genes, which have been acquired from the infected cell by recombination. Thus, retroviruses act as natural gene transfer vectors.
  • retroviral vectors useful for the transfer of many non- retroviral genes into a large variety of different cells
  • Miller, A. D. , Hum. Gene. Tiier. , 1 :5-14, 1990 retroviral vectors have been also used to transfer therapeutic genes into human cells to cure genetic diseases, cancer, AIDS, and various other diseases.
  • retroviral vectors used in clinical trials have been derived from murine leukemia virus (MLV), a C-type retrovirus, which is considered non- pathogenic for humans.
  • MLV has a rather simple genomic organization. It contains only two gene units, which code for the inner core structure proteins and the envelope protein, respectively. It does not contain regulatory genes like HIV-1.
  • the construction of safe gene delivery systems is rather simple and straightforward.
  • Such delivery systems consist of two components: the retroviral vector, which is a genetically modified viral genome that contains the gene of interest replacing retroviral protein coding sequences, and a helper cell that supplies the retroviral proteins for the encapsulation of the vector genome into retroviral particles ( Figure 1).
  • Modern helper cells contain separate plasmid constructs, which express all retroviral proteins necessary for replication ( Figure 1). After transfection of the vector genome into such helper cells, the vector genome is encapsulated into virus particles (due to the presence of specific encapsulation sequences) . Virus particles are released from the helper cell carrying a genome containing only the gene(s) of interest ( Figure 1). Thus, once established, retrovirus helper cells produce gene transfer particles for very long time periods (e.g. , several years). In the last decade, several retroviral vector systems have also been derived from other C-type retroviruses (Dornburg, R. , Gene Ther. , 2:301-310, 1995. ; Gunzburg, W. H. and
  • lentiviruses e.g. , HIV-1 or the simian immunodeficiency virus, SIV, which are able to establish a provirus in non-dividing cells.
  • lentiviruses e.g. , HIV-1 or the simian immunodeficiency virus, SIV
  • SIV simian immunodeficiency virus
  • Consensus nuclear translocation sequences have been identified in various proteins, which are located in the nucleus of eucaryotic cells. Such sequences are not 100% conserved, but are generally rich in the amino acids lysine, and arginine (see also Figure 2). It has been shown that the addition (insertion) of such sequences into various proteins enabled the transport of the modified proteins, which would normally reside in the cytoplasm, into the nucleus.
  • C-type retroviruses such as SNV do not contain a known nuclear translocation sequence.
  • the present invention introduces a nuclear translocation sequence into the SNV core protein, which enabled the virus particle to penetrate the nucleus of quiescent cells.
  • the present invention relates to a system by which therapeutic genes are transduced into quiescent cells, thereby allowing for a more efficacious treatment of genetic aberrations .
  • Plasmid constructs are indicated by the letter p (e.g. , pRD136) to distinguish them from the virus derived from the plasmid construct (e.g. RD136).
  • Plasmids The plasmid pCXL (Mikawa, T. , et al. , Exp. Cell Res. , 195:516-523, 1992). contains a SNV-derived retroviral vector expressing the bacterial ⁇ -galactosidase (LacZ) gene ( Figure 3). Plasmid pIM29 contains the complete wild-type envelope gene of SNV and has been described previously (Martinez, I. and Dornburg, R. , Virology, 208:234-241 , 1995). pRD136 is a gene expression vector, which contains the complete wild-type gag and pol genes of SNV 51.
  • Plasmid pTC53-7A5zeo is a gene expression vector derived from pTC53 33. It contains the zeocin gene, which mediates mammalian cell resistance to the antibiotic zeocin and expresses a chimeric single chain antibody (scA) - SNV envelope fusion protein ( Figure 3) to display antibody domains on the viral surface (Engelstadter, M. , et al. Targeting human T- cells by retroviral vectors displaying antibody domains selected from a phage display library, 1999 (submitted for publication).
  • scA chimeric single chain antibody
  • Figure 3 chimeric single chain antibody
  • the 7A5 scA has been selected from a phage display library and has been shown to bind to an antigen expressed on human T-lymphocytes and some other hematopoietic cell types (Engelstadter, M. , et al. Targeting human T-cells by retroviral vectors displaying antibody domains selected from a phage display library, 1999 (submitted for publication). Site-directed mutagenesis .
  • the SNV MA mutants were constructed by polymerase chain reaction (PCR) -mediated site-directed mutagenesis as described (Urban, A. , et al. , Nucleic Acids Res. , 25:2227-2228, 1997.;Picard, V. , et al. , Nucleic Acids Res. , 22:2587-2591, 1994.). Briefly, a 300 bp Eagl -Stul fragment from pRD136, which encodes the amino terminus of the SNV-MA protein, was cloned into the vector p-Alter vector purchased from Promega.
  • PCR polymerase chain reaction
  • the Eagl and Stul sites restriction enzyme sites were introduced in the outer PCR primers and were used for cloning the mutated DNA back into the pRD136 backbone digested with Eagl plus Stul .
  • the internal primers were designed to generate mutations as indicated ( Figure 2). After this back-cloning into pRD136, the plasmid DNAs were sequenced to verify the insertion of the correct mutation. After the introduction of the first mutation, the constructs containing single mutations were used as a template to create additional mutations.
  • D17 cells (a dog osteosarcoma cell-line) and the human cell lines 293T and HeLa (human cervical carcinoma) were obtained from the American Type Culture Collection ATCC, and were cultured in Dulbecco's modified Eagle's medium supplemented with 6% calf serum.
  • the human T-lymphoid cell lines Jurkat, C8166, and H9 were obtained from the courtesy of AIDS Reagent and Repository Program, National Institute of Allergy and Infectious Diseases (Bethesda). These cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 mg/ml and 2 mM L-glutamate.
  • Monocytes were obtained from the blood of a healthy human donor by centrifugation over Ficoll-Hypaque as described (Collman, R. , et al. , J. Exp. Med. ,
  • monocyte-derived macrophages MDM
  • monocytes were cultured at a cell concentration of 1 x 10 6 /ml in modified
  • MDM Dulbecco's medium
  • penicillin 100 U/ml
  • streptomycin 100 mg/ml
  • 10% pooled human serum at 37°C and 5 % CO 2 . Allowing the cells to adhere to the plastic surface without disturbance, monocytes differentiate into macrophages, which no longer undergo cell division (Collman, R. , et al. , J. Exp. Med. , 170: 1149-1163, 1989). After three weeks in tissue culture, the adherent macrophages were washed with medium to free them from remaining lymphocytes.
  • the protocol is similar to that described recently to test mutant envelope proteins (Martinez, I. and R. Dornburg, J. Virol , 70:6036-6043, 1996). Briefly, a stable cell line (derived from dog D17 cells) has been established which expresses the SNV envelope protein from plasmid pIM29 (Martinez, I. and Dornburg, R., Virology, 208:234-241 , 1995) and the retroviral vector pCXL, which transduces the bacterial ⁇ -galactosidase gene. (Mikawa, T. , et al. Exp. Cell Res. , 195:516-523, 1992). (Chu, T. -H. and R. Dornburg, J.
  • transfection virus 48 hours after transfection virus was harvested from confluent cultures and normal dividing as well as growth-arrested D17 cells were infected.
  • ⁇ Transient transfections were performed using the lipofectamine purchased from Bethesda Research Laboratories following the protocol recommended by the supplier. Briefly, for each transfection, 6 x 10 5 DSE29B-cxl cells were plated on a 60 mm diameter plastic dishes the day before transfection; 10 ⁇ g DNA was mixed in 15 ⁇ l lipofectamine. The cells were incubated with the DNA-lipofectamine mixture in 500 ⁇ l serum-free medium for 5 hrs.
  • D17 cells 2 ⁇ g/ml; human cells: 1 ⁇ g/ml), aphidicolin (5 ⁇ g/ml), mimocin (400 ⁇ M), or hydroxyurea (2.5 mM) as described 15,43.
  • mitomycin D17 cells: 2 ⁇ g/ml; human cells: 1 ⁇ g/ml
  • aphidicolin 5 ⁇ g/ml
  • mimocin 400 ⁇ M
  • hydroxyurea 2.5 mM
  • FACS Analysis of growth arrested cells Aliquots of 1 x 10 4 cells were fixed in 70% ethanol for 15 minutes followed by RNAase treatment (180 ⁇ g/ml) for 30 mins at RT. Next the DNA was stained with propidium iodide (50 ⁇ g/ml) at room temperature for 1 hr. Cells were evaluated by Fluorescence-activated cell sorter (FACS) analysis and the percentage of total viable cells in Gl , S, and G2/M phases of the cell-cycle was calculated by using the Cell-Fit software (Becton Dickinson).
  • FACS Fluorescence-activated cell sorter
  • the modification of the core protein should not dramatically impair protein folding, as an aberrantly folded protein may lead to impairment of virus core particle formation and/or function.
  • the nuclear translocation sequence should be at a similar position as that of a lentivirus, e.g. , HIV-1, which is located in the MA protein.
  • a lentivirus e.g. , HIV-1
  • many proteins of different species, which fulfil similar functions are folded in a very similar way, although their amino acid sequences can be considerably different.
  • the MA proteins of HIV-1 and SNV are folded in a similar way.
  • the introduction of a nuclear translocation sequence into MA of SNV at a position homologous to that of HIV-1 has the highest probability that the resulting genetically modified protein will function similarly to that of HIV-1.
  • This titer is approximately 1 ,000 fold lower than that obtained using a similar stabile transfected helper cell-line, e.g. , DSH-cxl cells (Dornburg, R., Gene Ther. , 2:301-310, 1995, Jiang, A. , et al. , J. Virol , 72: 10148- 10156, 1998), and is comparable to titers obtained in other previous transient transfection / infection experiments (Chu, T.-H. and Dornburg, R., J. Virol , 69:2659-2663, 1995), using the components of the SNV vector system described in the present invention.
  • DSH-cxl cells Dornburg, R., Gene Ther. , 2:301-310, 1995, Jiang, A. , et al. , J. Virol , 72: 10148- 10156, 1998)
  • SNV vector particles with wt-SNV envelope proteins are not infectious in human cells (Dornburg, R. , Gene Ther. , 2:301-310, 1995); Chu, T.-H. and Dornburg, R. J. Virol , 69:2659-2663, 1995; Jiang, A., et al., J. Virol , 72: 10148- 10156, 1998; Blaese, R.M. , et al., Science, 270:475-480, 1995).
  • a highly efficient, cell-type-specific gene transfer into human cells can be obtained, when a targeting envelope is co-present with wild-type Env (Chu, T.-H. and Dornburg, R. , J.
  • transfection into DSE29B-cxl cells was performed with the Gag-Pol expression vectors plus a plasmid expressing the scA-Env chimeric fusion protein (pTC53-7A5-zeo, Figures 3 and 4).
  • the scA recognizes an antigen present on human T-lymphocytes and other hematopoietic cells.
  • the packaging lines produce vector particles with two envelopes capable of infecting human T-lymphocytes with high efficiency (Engelstadter, M. , et al. Targeting human T-cells by retroviral vectors displaying antibody domains selected from a phage display library, 1999 (submitted for publication)).
  • a vector virus titer of 5 x 10 2 was approximately 4, 000-fold lower than that obtained with a stable packaging cell-line, which expressed the lacZ vector and the wild-type viral particle proteins from the same gene expression plasmids.
  • This much lower titer reflects the transient double-transfection protocol, in which only a small percentage of the transfected helper cells express relatively low amounts of all viral proteins necessary for complete virus particle assembly.
  • Virus particles containing the wild-type MA and targeting Env were not able to infect any of the T-cell-lines which were growth-arrested with mitomycin.
  • RD136-m7 was able to significantly infect such growth-arrested cells (Table 3). These data confirm the results obtained in dog D17 cells and show that the nuclear translocation signal sequences in the SNV-MA is sufficient to allow infection of growth-arrested human cells. To test this further, a stable cell-line was created, which continuously expresses all retroviral particle proteins. Briefly, DSE29B-cxl cells were transfected with pRD136-m7 in co-transfection with plasmid pTC53-7A5zeo, followed by zeocin selection.
  • zeocin resistant colonies Five individual zeocin resistant colonies were isolated and cell-lines were established from such clones. All clones were briefly tested for their ability to transduce the lacZ gene into dividing human T-lymphocytes and the clone with the highest gene transduction efficiency was selected for further experiments.
  • Virus was harvested from confluent tissue culture plates and infectivity experiments were performed with dividing and growth-arrested human T- lymphocytes, as described above. T-lymphocytes were infected with serial virus dilutions and the number of lacZ-transduced cells was determined. Dividing C8166 cells were infected with a titer of 6 x 10 5 cfu/ml.
  • DSE29B-cxl cells contain transfected copies of the retroviral vector pCXL. PCR amplification of this control DNA resulted in a DNA band which migrated in an agarose gel at the expected size. An additional smaller band was also seen, which was the result of a non-specific PCR amplification ( Figure 6). No DNA bands were seen in control experiments with DNA isolated from the nuclei of uninfected D17 cells or H9 cells ( Figure 6). These data show that the PCR- amplified fragment was specific for the lacZ gene. Bands specific for the lacZ gene were obtained from DNAs isolated from nuclei of dividing cells infected with wild- type vectors ( Figure 6).
  • lacZ PCR products were obtained from DNAs of growth-arrested H9 or D17 cells, respectively, infected with wild-type vectors.
  • lacZ specific DNA was obtained from DNA of growth arrested D17, H9, or Jurkat cells infected with vectors containing an nuclear translocation signal sequence.
  • monocyte-derived macrophages blood-derived monocytes were allowed to adhere to the plastic surface of tissue culture dishes without disturbance and cultivated for three weeks. After this time period, aliquots of the cells were subjected to FACS analysis. This analysis confirmed the complete quiescent state of such cells. The rest of the cells were infected with vector virus harvested from four different packaging lines. As expected, vector virus particles containing wt-MA and wt-Env only, did not infect quiescent macrophages at all ( Figure 7A).
  • retroviral vectors derived from C-type viruses such as MLV
  • Lentiviruses such as HIV-1 or simian immunodeficiency virus (SIV-1) are able to infect quiescent cells.
  • SIV-1 simian immunodeficiency virus
  • the introduction of a nuclear localization sequence into the MA protein of SNV is sufficient to trigger the active import of the SNV pre-integration complex into the nucleus of an infected cell.
  • Introducing the nuclear localization sequence in the SNV-MA protein at a position homologous to that in HIV-1 will have the highest chance of success for the following reason: it is widely accepted that all retroviruses evolved from a common ancestor millions of years ago (Temin, H.M. , J. Natl. Cancer hist. , 46:3-7, 1971 ; Coffin, J.M. , Curr. Top. Microbiol. Immunol , 176: 143-164, 1992).
  • introducing the nuclear translocation signal sequence at a position similar to that in HIV-1 w ould expose the introduced nuclear translocation signal sequence as an endogenous motif and, therefore, would be recognized by cellular protein complexes involved in the nuclear import of proteins or protein complexes.
  • a series of different mutations were introduced into the MA protein of SNV.
  • SNV revealed a similar sequence. That is, two lysine residues, which are an essential part of nuclear translocation signal sequence, were already present (position 2 and 3).
  • SNV contained an arginine residue.
  • Arginine seems to be exchangeable with lysine, since different HIV-1 isolates contain either arginine or lysine at the sixth position ( Figure 2). Thus, it appeared that a maximum of three amino acid exchanges were necessary to introduce a nuclear translocation signal sequence into the SNV-MA. To test whether any of the mutations had a negative effect on viral replication, and to determine, whether the mutation(s) would endow the virus particle with the ability to actively enter the nucleus of an infected cell, a series of mutants was created and investigated individually. All mutations were tested in a transient transfection / infection assay system. The disadvantage of this assay system is that only relatively low vector virus titers can be obtained.
  • a titer of 10 3 cfu/ml appears to be the upper limit using the plasmids described and dog D17 cells (30,32,34).
  • the main advantage of this system is that virus titers represent a statistically significant average virus titer produced from thousands of transfected cells.
  • the spontaneous production of replication-competent virus has never been observed using the components of this vector system (Martinez, I. and Dornburg, R. , Hum. Gene Ther. , 7:705-712, 1996). This allowed the direct comparison of the biological activity of the different constructs. Using this experimental system, wild-type SNV was completely unable to infect quiescent cells.
  • the introduction of a single mutation which had no effect on normal viral replication, was also not sufficient to enable the infection of quiescent cells.
  • the introduction of at least two mutations endowed the MA protein with a viable nuclear translocation signal sequence.
  • one mutant, RD136-m7 which contained only two substitutions at amino acid positions 5 and 6 (28 and 29 in the A A sequence), consistently revealed the highest infectivity in all experiments, and gave slightly higher vector virus titers than a mutant containing the complete HIV-1 consensus nuclear translocation signal sequence. Even further, the efficiency of infection in dividing cells of this double mutant was reproducible higher than that of wt-SNV.
  • Viral titers of a few hundred to one thousand cfu/ml appear to be low in comparison to titers obtained from comparable stable packaging lines (10 6 cfu/ml), which had been selected from a single high producer clone (Engelstadter, M., et al. , Targeting human T-cells by retroviral vectors displaying antibody domains selected from a phage display library, 1999 (submitted for publication)).
  • reproducible virus titers ranging between 100 to 1 ,000 cfu/ml are still high enough to allow conclusions, in particular, because the background infectivity of control constructs consistently remained at undetectable levels ( ⁇ 1 cfu/ml).
  • virus titers in growth-arrested cells were slightly lower than that obtained in dividing cells.
  • this decrease in titer also reflects the biological condition of growth- arrested cells, since many cells undergo apoptosis, i.e: programmed cell death, during the growth-arrest imposed by the inhibitors of mitosis, a very well known observation (Bukrinsky, M.I. , et al. , Nature, 365:666-670, 1993; Lewis, P. and Emerman, M., J. Virol , 68:510-516, 1994; Schwedler, U. , et al. , Proc. Natl. Acad. Sci.
  • virus titers observed (up to 500 over a background of no infected cells in control experiments) clearly show that the SNV with a nuclear translocation signal sequence is able to infect non-dividing cells.
  • initial experiments indicate that more than 1 , 000-fold higher virus titers can be obtained in quiescent T-lymphocytes and macrophages using vector virus harvested from a stable packaging line.
  • even higher titers may be obtained be screening and selecting a single high producer clone from a large number of isolated cell clones.
  • Virus particles were harvested from DSE29B-xcl cells transfected with plasmids encoding SNV gag-pol proteins (wt or mutant) and dividing or growth-arrested D17 cells were infected.
  • Virus titers expressed as colony forming units per ml supernatant medium (cfu/ml), were determined 48 hours after infection by counting clue cell colonies (2 or 4 adjacent cells), or in the case of growth-arrested single blue cells. The titers presented here are avaraged from three independent experiments.
  • Virus particles were harvested from DSE29B-cxl cells transfected with plasmids encoding SNV gag-pol proteins (wt or mutant) and radiated (r) or non-radiated (nr), growth-arrested D17 cells were infected.
  • Virus titers expressed as colony forming units per ml supernatant medium (cfu/ml), were determined 48 hours after infection by counting blue cells. The titers presented here are averaged from three independent experiments. Cells were radiated before or after the inhibitor of mitosis was added and no difference in virus titer was observed.
  • Table 3 Virus was harvested from DSE29B-cxl cells transiently transfected with SNV- gag-pol expression vectors plus a plasmid for the expression of a targeting envelope (RD136 (wt) and RD136-m7).
  • the targeting envelope was a chime ⁇ c scA-TM protein.
  • the scA (termed 7A5) recognizes an antigen present on human T-lymphocytes and macrophages.
  • Infectivity experiments were performed in the human T-cell lines indicated. Growth-arrest was induced with mitomycin.
  • the data presented are the average of two sets of independent experiments, in which very similar results were achieved.
  • the bootom row shows virus titers obtained from a stable packaging line (RD136-ml-stable) transfected with pRD136-m7 and pTC53-7A5zeo. nd: not done.

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Abstract

L'invention concerne une technique de production de particules de vecteur rétroviral dérivées de rétrovirus, et capables de transduire des gènes thérapeutiques en cellules ne se divisant pas. Les mutations de la protéine à matrice (MA) rétrovirale permettent l'infection desdites cellules ne se divisant pas.
PCT/US2000/002852 1999-02-02 2000-02-02 Particules de vecteur retroviral mises au point par genie genetique, capables d'infecter des cellules ne se divisant pas WO2000046377A1 (fr)

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US20030087264A1 (en) * 2001-05-22 2003-05-08 Kaplitt Michael G. Transcriptional regulation of target genes
US20040038304A1 (en) * 2002-03-28 2004-02-26 Gala Design, Inc. Antibody libraries

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